Gamma-hydroxy butyric acid (4-hydroxybutanoic acid, C4H8O3), commonly abbreviated GHB, is an endogenous substance and a therapeutic drug which is illegal in multiple countries and a naturally occurring substance found in the central nervous system, vine, beef, small citrus fruits and almost all other living creatures in small amounts. It is currently regulated in the US and sold by Jazz Pharmaceuticals under the name Xyrem.
In a medical setting, GHB is used as a general anesthetic, to treat conditions such as insomnia, clinical depression, narcolepsy, and alcoholism, and to improve athletic performance.
In Italy, GHB is used in the treatment of alcoholism (50 to 100 mg per kg per day in three or more divided doses) under the trade name Alcover (ATCN 07 BB), both for acute alcohol withdrawal and medium to long term detoxification. In the United States, the Food and Drug Administration (FDA) permits the use of GHB under the trade name Xyrem to reduce the number of cataplexy attacks in patients with narcolepsy.
When GHB is used in its sodium or potassium salt form, a significant quantity of excess sodium or potassium may be consumed, which should be taken into consideration by people with heart insufficiency, hypertension or reduced renal function. The bioavailability of sodium GHB is considerably reduced when it is consumed with food, and so it is advised to wait at least two hours after eating before consuming the dose. Because of its strong sedative effects, patient should not drive or operate heavy machinery for at least six hours after taking sodium GHB.
Adverse effects from Xyrem in clinical trials included: headache, nausea, nasopharyngitis, dizziness, somnolence, vomiting, urinary incontinence, confusion, dyspnea, hypoesthesia, paresthesia, tremor, vertigo, and blurred vision.
Gamma-hydroxy butyrate (GHB) is also an illicit chemical that has become a major cause of drug related comas in the US and other countries. In fact, the number of GHB overdoses in the United States has now out-paced overdoses from MDMA (Ecstasy). GHB was rejected by the American medical community in the 1960s, but has become popular among many people for its ability to cross the blood brain barrier freely and depress consciousness, resulting in euphoria, but also toxic effects. It is also touted on the Internet as a sleep aid and anti-depressant and weight loss product although these uses are not substantiated by proven scientific studies and may carry potentially deadly twists. Starting first as an alternative to steroids in the late 1980s when steroids were being controlled, GHB has grown into a multiheaded medical nightmare draining emergency room services, shattering the lives of those who have lost loved ones to it and terrifying families/friends of those addicted to it. Yet, it is still a mystery to most law enforcement officers, medical/coroner personnel and parents.
Non-medically, GHB also acting as a central nervous system (CNS) depressant, is used as a drug of abuse. It has many street names including Liquid Ecstasy and Liquid X. At low doses, GHB can cause a state of euphoria, increased enjoyment of movement and music, increased libido and increased sociability. At higher doses, GHB my induce nausea, dizziness, drowsiness, agitation, visual disturbances, depressed breathing, amnesia, unconsciousness, and death. The effects of GHB can last from 1.5 to 3 hours or even longer, if large doses were consumed or if it was mixed with alcohol.
In general, the doses used recreationally are between 500 mg and 3000 mg, corresponding to approximately 0.5 to 3 ml of liquid if the concentration is 1 g/ml (which is not always the case). When used as a recreational drug, GHB may be found as a pure liquid or as GHB salt dissolved in water generally at a standardised concentration of 1 g/ml and so it is twice the strength of the drug, Xyrem, sold legally for medical use.
GHB salt dissolved in water and/or alcoholic beverages is notoriously dangerous as the concentration of GHB may not be known and so the actual dose of GHB being consumed can be difficult to judge accurately. Since GHB sold for recreational use is subject to no standardisation it can be impossible to verify the actual concentration of GHB solution bought on the illicit market. More than 1 g of sodium GHB can be dissolved in 1 ml of water and so sodium GHB solution can actually be stronger than pure GHB liquid. Other salt forms such as potassium GHB, calcium GHB and magnesium GHB have also been reported but the sodium salt is by far the most common.
Some chemicals are converted to GHB in the stomach and when circulating in the bloodstream. GBL or gamma-butyrolactone is one of such prodrugs. Other prodrugs include 1,4-butanediol (1,4-B). There may be additional toxicity concerning these precursors. 1,4-B and GBL are normally found as pure liquids, although they may be mixed with other more harmful solvents when intended for industrial use, e.g. as paint stripper or varnish thinner.
GHB can be produced in clandestine labs and it is claimed that most of the GHB used in the US is illegally manufactured within its borders. While available as a prescription for sleep disorders in some other countries, GHB was banned (in the US) by the FDA in 1990 because of the dangers associated with its use. However, on Jul. 17, 2002, GHB was approved for treatment of cataplexy often associated with narcolepsy.
GHB was first synthesised in France more than 40 years ago as a possible anesthetic but because of its undesirable side effects was rejected by the American medical community. Its legal use anywhere is dwindling as countries are beginning to recognize the problems. GHB resurfaced in 1987 as an orphan drug being researched to treat the combination of sleep disorders known as narcolepsy/cataplexy. At about the same time, steroid users were told that it might enhance the body's production of growth hormones (in-deep-sleep-state). However, due to growing numbers of overdoses, it was ordered off the shelves of stores in November 1990. Unfortunately, it has gained status as a recreational drug and as a rape drug and has become dangerously common. As a result of increased restrictions on GHB itself, its ‘analogs’ or chemical relevants that can be converted to GHB in the body, have become increasingly prevalent.
The action of GHB has yet to be fully elucidated. GHB clearly has at least two sides of action, stimulating the latey characterized and aptly named “GHB receptor” as well as the GABAB receptor. GHB, if it is indeed a neurotransmitter, will normally only reach concentrations high enough to act at the GHB receptor as it has relatively weak affinity for GABAB receptor. However, during recreational usage, GHB can reach very high concentrations in the brain, relative to basal levels and can act at the GABAB receptor as well. The action of GHB at the GABAB receptor are probably responsible for its sedative effects. GHB-mediated GABAB receptor stimulation inhibits dopamine release as well as causes the release of natural sedative neurosteroids (like other GABAB agonists such as Baclofen). In animals, the sedative effects of GHB can be stopped by GABAB antagonists (blockers).
The relevance of the GHB receptor in the behavioural effects induced by GHB is more controversial. It seems hard to believe, that the GHB receptor is not important when it is densely expressed in many areas of the brain, including the cortex, as well as it being the high affinity site of GHB action. There has only been limited research into the GHB receptor. However, there are evidences that it causes the release of glutamate which is a stimulatory neurotransmitter. Drugs which selectively activate the GHB receptor but not the GABAB receptor such as trans-4-hydroxycrotonic acid and 4-(p-chlorobenzyl)-GHB cause convulsions in animals and do not produce GHB-appropriate responding.
Activation of the GHB receptor does not alone explain GHB's addictive properties; research using selective GABAB agonists and analogues of GHB, which are selective agonists for the GHB receptor but do not activate GABAB, suggested both the GHB receptor and the GABAB receptor are important for dopamine release and consequently abuse liability. Compounds which activate only one of the receptors but not both, do not seem to induce acute dopamine release or to exert the abuse potential typical for GHB itself.
Generally high doses of GHB are sedative through its action at the GABAB receptor, while lower doses are stimulatory through activation of GHB receptors. This may explain the paradoxical mix of sedative and stimulatory properties typical for GHB intoxication, as well as the so called “rebound” effect, experienced by individuals using GBH as a sleeping agent, when they awake suddenly after several hours of GHB-induced deep sleep. This is due to the fact, that the concentration of GHB in the system decreases because of metabolism below a threshold for stimulating GABAB receptor function, and then stimulates the GHB receptor leading to wakefulness.
The depressant effects of GHB on the brain in low doses produce a high or euphoric feeling as inhibitions are depressed. When the dose is increased, profound coma results. The heart rate may also be depressed or slowed down. Effects on the nervous system may result in a spasm of muscle contractions called myoclonus, producing seizure-like movements. Other effects such as confusion, amnesia, vomiting and irregular breathing are dangerous when combined with the major depressant effects of GHB. Other drugs in combination with GHB, particularly alcohol, may worsen the depressive effect and increase the possibility of a fatal outcome. The desired effects for GHB in low doses may sound inviting, but the consequence of a (falsely) high dose may be death. The dosage response of GHB is quite steep, meaning that a tiny increase in dose may cause a dramatic increase in symptoms and risk. Variable effects mean that a teaspoon might be perfect one time, but may become an overdose the next time. It is also important to be aware of the consequences that occur when GHB is mixed with other chemicals. For instance, mixing GHB with alcohol or other depressants is even more likely to result in death. The effects last about four hours and can resolve quite suddenly.
The drug has furthermore recently been referred in the media to as a date rape drug, in much the same way as alcohol and the drug, Rohypnol. GHB by itself has a soapy or salty taste, but diluted in solutions it is almost tasteless and, therefore, when mixed in some drinks it is difficult to detect. Moreover, identification of GHB after consumption is complicated by the short duration of time that it persists in body fluids. So far, several testing methods have been developed within the art: methods for screening of gammy-hydroxybutyric acid (GHB) in body fluids or hair but also in beverages.
Kintz et al. described for example a testing method for GHB in hair by gas chromatography (GC)/mass spectroscopy (MS). The method requires decontamination of the hair sample with dichloromethane followed by overnight incubation in 0.01 N NaOH in the presence of GHB-d6, followed by neutralisation and extraction in ethyl acetate under acidic conditions (J. Forensic Sci., 2003, January; 48(1): 195-200), which is incorporated by reference. A similar method has been developed by Shen et al. (Fa Yi Xue Za Zhi; 2006 February; 22(1): 48-51), which is incorporated by reference who provide a GC/MS assay for GHB in hair. Both methods do not only have the disadvantage that GC/MS testing methods are time consuming and elaborate, but also that the hair sample can soonest be collected one month after the alleged event in order to sample the corresponding period of the regular growing. Therefore, real-time and onsite testing is not possible.
Until now, ways to measure GHB in blood and urine have been almost limited to chromatographic methods, such as GC-MS, LC/LC-MS, HPLC, HPLC-MS or capillary electrophoresis (CE).
Testing methods for determination of GHB in blood and urine samples were for example developed by Kankaanpaa et al. (Forensic Sci Int. 2007 Aug. 6; 170 (2-3): 133-8), which is incorporated by reference. This method also uses a GC/MS analysis after several extractions, acidification and centrifugation steps. Similar methods (GC/MS in association with several conditioning and/or preparation steps) are provided by Liu et al. (Fa Yi Xue Za Zhi; 2007 April; 23(2): 120-2, 129), which is incorporated by reference, Paul et al. (J Anal Toxicol. 2007 July-August; 30(6): 375-9), which is incorporated by reference, Ferrara et al. (J Farm Biomed Anal. 1993 June; 11(6): 483-7), which is incorporated by reference, Villain et al. (J Chromatogr B Analyt Technol Biomed Life Sci. 2003 Jul. 15; 792(1): 83-7), which is incorporated by reference, McCusker et al. (J Anal Toxicol. 1999 September; 23(5): 301-5), which is incorporated by reference, Elian (Forensic Sci Int. 2000 Apr. 10; 109(3): 183-7), which is incorporated by reference and Blanchet et al. (J Chromatogr B Analyt Technol Biomed Life Sci. 2002 Apr. 5; 769(2): 221-6), which is incorporated by reference. Gas-chromatographic (GC) methods were also commonly used for the detection of GHB in beverages and/or drinks (e.g. Liu et al., Fa Yi Xue Za Zhi, 2007 April; 23(2): 120-2, 129), which is incorporated by reference.
Further testing methods for GHB content include a colour test for rapid screening of GHB in drinks and urine characterized in that GHB was converted into an acidic solution to GBL which reacted with hydroxylamine hydrochloride in presence of sodiumhydroxide forming hydroxamate. A purple complex was formed when hydroxamate reacted with ferric chloride in acidic condition (Zhang et al.; Fa Yi Xue Za Zhi, 2006 December; 22(6): 424-7), which is incorporated by reference.
Further, a method of determination of GHB in human urine by capillary electrophoresis (CE) with indirect UV detection and confirmation with electrospray ionisation ion-trap mass spectrometry is known within the art (Baldacci et al., J Chromatogr A, 2003 Mar. 21; 990(1)-2: 99-110), which is incorporated by reference. The assay is based on liquid extraction and capillary zone electrophoresis (CZE) with indirect UV absorption detection. The background electrolyte is composed of 4 mM nicotinic acid (compound for indirect detection), 3 mM spermine (reversal of electro-osmosis) and histidine (added to reach a pH of 6.2). Having a 50 micron I.D. capillary of 40 cm effective length, 1-octanesulfonic acid as internal standard, solute detection at 214 nm and a diluted urine with a conductivity of 2.4 mS/cm, GHB concentrations ≧2 μg/ml can be detected.
In addition, an enzyme based assay for GHB detection has been developed by Bravo et al., wherein the GHB content of a sample is determined in a two step testing method using GHB dehydrogenase from Ralstonia eutropha. This method consists of at least two consecutive steps, wherein the first step consists in contacting the sample with the GHB-oxidoreductase and an oxidized cofactor resulting in the reduction of the oxidized cofactor and wherein the second step consists in contacting the sample and the reduced cofactor with a second oxidoreductase and a chromogen or dye resulting in the formation of a detectable compound (Bravo et al., J Forensic Sci, March 2004, Vol. 49, No. 2: 379-87), which is incorporated by reference. This method is further patented by U.S. Pat. No. 6,703,216 B2, which is incorporated by reference.
All of the methods already known within the state of the art require either extensive equipment, and/or highly skilled personnel and/or take a long time to conduct. The need for a new, simple and rapid method which can also be accomplished by less qualified laboratory personnel, does not require extensive equipment to conduct and—in addition very fast—making the method also suitable for emergency testing.